: Viral pathogens remain a major public health threat, and their persistence is largely due to the mechanisms they have adapted to evade or combat our immune systems. Cellular defense proteins called restriction factors are important for stifling the initial spread of infection both within and between people, but many viruses have evolved proteins capable of antagonizing them. The protein surfaces used by many viral proteins to bind and inactivate restriction factors are not known, making it difficult to disrupt them to promote immunity. Mutagenesis experiments can address these gaps in our knowledge, but conventional methods are too slow and laborious to test the many thousands of possibly informative protein mutants that exist. I propose to use a new experimental approach called deep mutational scanning to study how all of the possible single amino acid variants of a viral immune evasion protein or host restriction factor affects the ability of a virus to infect cells. In the first aim, I will test thousands of protein variants of the HIV-1 protein Vif to figure out which amino acids it uses to bind and neutralize each of the different APOBEC3 restriction factors. This will help us understand how Vif has evolved to keep binding each APOBEC3 protein without losing binding against the others. This work will also help us understand and improve upon antivirals that target Vif. In the second aim, I will assay the activity of thousands of variants of the restriction factor Tetherin in the presence of diverse viral proteins that have evolved to neutralize it. By comprehensively identifying Tetherin variants capable of escaping each antagonist, I will infer each of their binding sites. By comparing the relative strength and number of possible escape variants for each antagonist, I will learn which mechanisms are most effective in neutralizing Tetherin. This work will reveal discrete proteins interaction surfaces that may be disrupted by antiviral drugs. These results will also reveal successful strategies viruses have evolved to inhibit immune proteins, which will make us better able to find new ways to stop them. This project will highlight the power of using deep mutational scanning to gain broad, unbiased views into the mechanisms that viruses have evolved to neutralize our immune systems, which I plan to expand upon in my research lab.